The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules

BACKGROUND: Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11–12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. RESULTS: Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. CONCLUSION: Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model”

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model

The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules

Abstract Background Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11–12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. Results Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. Conclusion Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily.</p

ERBB4 mutations in cancer and amyotrophic lateral sclerosis

ErbB receptor tyrosine kinases, epidermal growth factor receptor (EGFR, also known as ErbB1), ErbB2 (HER2 or NEU), ErbB3 (HER3), and ErbB4 (HER4), transduce signals borne by extracellular ligands into central cellular responses such as proliferation, survival, differentiation, and apoptosis. Mutations in ERBB genes are frequently detected in human malignant diseases of epithelial and neural origin, making ErbB receptors important drug targets. Targeting EGFR and ErbB2 has been successful in eg. lung and breast cancer, respectively, and mutations in these genes can be used to select patients that are responsive to the targeted treatment. Although somatic ERBB4 mutations have been found in many high-incidence cancers such as melanoma, lung cancer, and colorectal cancer and germ-line ERBB4 mutations have been linked to neuronal disorders and cancer, ErbB4 has generally been neglected as a potential drug target. Thus, the consequences of ERBB4 mutations on ErbB4 biology are largely unknown. This thesis aimed to elucidate the functional consequences and assess the clinical significance of somatic and germ-line ERBB4 mutations in the context of cancer and amyotrophic lateral sclerosis. The results of this study indicated that cancer-associated ERBB4 mutations can promote aberrant ErbB4 function by activating the receptor or inducing qualitative changes in ErbB4 signaling. ERBB4 mutations increased survival or decreased differentiation in vitro, suggesting that ERBB4 mutations can be oncogenic. Importantly, the potentially oncogenic mutations were located in various subdomains in ErbB4, possibly providing explanation for the characteristic scattered pattern of mutations in ERBB4. This study also demonstrated that hereditary variation in ERBB4 gene can have a significant effect on the prognosis of breast cancer. In addition, it was shown that hereditary or de novo germ-line ERBB4 mutations that predispose to amyotrophic lateral sclerosis inhibit ErbB4 activity. Together, these results suggest that ErbB4 should be considered as a novel drug target in cancer and amyotrophic lateral sclerosis.ERBB4-geenin mutaatiot syövässä ja amyotrofisessa lateraaliskleroosissa ErbB reseptorit, epidermaalisen kasvutekijän reseptori (EGFR tai ErbB1), ErbB2 (HER2 tai NEU), ErbB3 (HER3) ja ErbB4 (HER4), välittävät solun ulkopuolisten kasvutekijöiden tuomia signaaleja solun sisään, ja siten säätelevät keskeisiä solun toimintoja kuten kasvua, selviytymistä, erilaistumista ja kuolemaa. ERBB-geenien mutaatioita havaitaan toistuvasti eri syövissä. ErbB-reseptorit ovat merkittäviä lääkehoidon kohdemolekyylejä. Mutatoituneisiin EGFR- ja ErbB2-reseptoreihin kohdistettuja lääkehoitoja käytetään mm. keuhko- ja rintasyöpäpotilaiden hoidossa. Somaattisia ERBB4-geenin mutaatioita on havaittu useissa yleisissä syövissä kuten melanoomassa, keuhkosyövässä sekä paksu- ja peräsuolisyövässä. ERBB4-geenistä on tunnistettu myös monia ituradan mukana periytyviä mutaatioita keskushermoston sairauksissa ja syövissä. ErbB4-reseptorin merkitys lääkehoidon kohteena tunnetaan kuitenkin huonosti. Tässä tutkimuksessa pyrittiin selvittämään, miten ERBB4-geenin mutaatiot vaikuttavat reseptorin toimintaan, sekä arvioimaan ERBB4-geenin mutaatioiden kliinistä merkitystä syövässä ja amyotrofisessa lateraaliskleroosissa. Tutkimuksen tulokset osoittavat, että syövässä esiintyvät ERBB4 geenin mutaatiot voivat johtaa epänormaaliin reseptorin toimintaan joko aktivoimalla reseptorin, tai aiheuttamalla muutoksia reseptorin solunsisäisessä viestinvälityksessä. Nämä muutokset johtivat lisääntyneeseen syöpäsolujen selviytymiseen tai vähentyneeseen solujen erilaistumiseen in vitro. Tulosten perusteella ErbB4-reseptorin mutaatiot voivat olla onkogeenisiä. Huomionarvoista on, että nämä mahdollisesti onkogeeniset mutaatiot olivat jakaantuneet useaan ErbB4-reseptorin rakenteelliseen alayksiköön. Tutkimuksessa osoitetiin myös, että ERBB4-geenin perinnöllinen variaatio voi vaikuttaa rintasyöpäpotilaan taudin ennusteeseen. Lisäksi tutkimuksessa näytettiin, että ERBB4 geenin joko perinnölliset tai de novo ituradan mutaatiot, jotka altistavat amyotrofiselle lateraaliskleroosille, vähentävät ErbB4-reseptorin aktiivisuutta. Tulosten perusteella mutatoitunut ErbB4-reseptori voi olla potentiaalinen lääkehoidon kohde syövässä ja amyotrofisessaSiirretty Doriast

Simultaneous Inhibition of ErbB3 and Calmodulin-Mediated Signaling Effectively Inhibits Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival

Patients with Neurofibromatosis Type 1 have germline mutations in the neurofibromin gene (NF1) and are prone to develop tumors in the nervous system and elsewhere throughout their lifespan. Loss-of-function mutations of the remaining functional copy of the NF1 tumor suppressor gene in the Schwann cell lineage results in development of benign tumors known as dermal and plexiform neurofibromas. Mutations in additional tumor suppressor genes, like p53 and CDKN2A, subsequently transforms plexiform neurofibromas into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). At present, no effective treatments are available for MPNSTs, in fact some approaches make these tumors more aggressive. Since both neurofibromas and MPNSTs demonstrate Ras hyperactivation, many laboratories have targeted Ras and key downstream effectors of Ras. Unfortunately, all attempts to develop downstream targeted therapies for MPNSTs have failed. However, upstream receptor tyrosine kinases (RTKs) can activate Ras through several mechanisms in MPNSTs. One RTK, erbB3, has yet to be investigated in MPNST pathogenesis. Furthermore, upstream, and parallel Ras activation by intracellular calcium signaling has also been implicated in MPNSTs but has not yet been investigated. We found that erbB3 is expressed and required for the proliferation and survival of MPNST cells and that erbB3 promotes calcium-mediated signaling viii mechanisms. Using genome-wide shRNA screens, NRG1β-erbB3 microarray analyses and in vitro drug kinomics screening, we identified novel erbB3 and calcium regulated signaling pathways as potential druggable targets for MPNSTs. Due to the historical failure of monotherapy drug treatments in MPNST patients, we sought to identify a combinatorial treatment that effectively inhibited MPNST cell proliferation and survival by targeting upstream activators. We discovered that erbB3 signals through both canonical and novel pathways in MPNSTs and that calcium-calmodulin mediated signaling is an MPNST vulnerability. We have identified a novel calmodulin-independent regulation of the calmodulin effector calmodulin-regulated kinase II (CamKII) and have shown that simultaneous inhibition of erbB3 and calcium-calmodulin mediated signaling significantly reduces MPNST cell proliferation and survival. We conclude that erbB3 contributes significantly to MPNST growth and survival as well as calcium-calmodulin mediated signaling and that inhibition of both pathways is more effective than treatment with erbB or calmodulin inhibitors alone

Role of ErbB2 and ErbB4 in Cancer Growth, Prognosis and as Targets for Immunotherapy

ErbB receptors (EGFR, ErbB2, ErbB3 and ErbB4) are growth factor receptors that regulate signals of cell differentiation, proliferation, migration and survival. Inappropriate activation of these receptors is associated with the development and severity of many cancers and has prognostic and predictive value in cancer therapy. Drugs, such as therapeutic antibodies, targeted against EGFR and ErbB2, are currently used in therapy of breast, colorectal and head and neck cancers. The role of ErbB4 in tumorigenesis has remained relatively poorly understood. Alternative splicing produces four different isoforms of one ErbB4 gene. These isoforms (JM-a, JM-b, CYT-1 and CYT-2) are functionally dissimilar and proposed to have different roles in carcinogenesis. The juxtamembrane form JM-a undergoes regulated intramembrane proteolysis producing a soluble receptor ectodomain and an intracellular domain that translocates into the nucleus and regulates transcription. Nuclear signaling via JM-a isoform stimulates cancer cell proliferation. This study aimed to develop antibodies targeting the proposed oncogenic ErbB4 JM-a isoform that show potential in inhibiting ErbB4 dependent tumorigenesis. Also, the clinical relevance of ErbB4 shedding in cancer was studied. The currently used monoclonal antibody trastuzumab, targeting ErbB2, has shown efficacy in breast cancer therapy. In this study novel tissues with ErbB2 amplification and trastuzumab sensitivity were analyzed. The results of this study indicated that a subpopulation of breast cancer patients demonstrate increased shedding and cleavage of ErbB4. A JM-a isoform-specific antibody that inhibited ErbB4 shedding and consequent activation of ErbB4 had anti-tumor activity both in vitro and in vivo. Thus, ErbB4 shedding associates with tumor growth and specific targeting of the cleavable JM-a isoform could be considered as a strategy for developing novel ErbB-based cancer drugs. In addition, it was demonstrated that ErbB2 amplification is common in intestinal type gastric cancers with poor clinical outcome. Trastuzumab inhibited growth of gastric and breast cancer cells with equal efficacy. Thus, ErbB2 may be a useful target in gastric cancer.Siirretty Doriast

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.Siirretty Doriast

Characterizing and engineering antibodies against the epidermal growth factor receptor

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Vita.Includes bibliographical references.Epidermal growth factor receptor (EGFR) signaling leads to cellular proliferation and migration, and thus EGFR dysregulation can significantly contribute to the survival of tumor cells. Aberrant EGFR signaling due to receptor overexpression, mutation, or autocrine ligand has been observed in a wide variety of malignancies, and antibody drugs which inhibit EGFR signaling have been developed. However, the epitopes of most EGFR antibodies have not been characterized, and the marginal efficacies of current antibodies underscore the need for improved therapeutics. In this thesis work, we have created a novel method of epitope mapping, which is the determination of antigen residues responsible for mediating an antibody-antigen interaction. In our technique, a library of random mutants of the EGFR antigen is displayed on the surface of yeast, and the library is combinatorially selected for loss of binding to the antibody being mapped. If a mutant shows loss of binding to an antibody, then that residue is a potential contact residue. In addition, we found that many mutants caused a global misfolding of the antigen, requiring the use of high-throughput sorting to remove the misfolded mutants. The development of our epitope mapping method using random mutagenesis and yeast surface display enabled the successful mapping of four different antibodies and three designed ankyrin repeat proteins binding to EGFR. In addition, we continued work on engineering novel antibodies against EGFR domains II and IV. Antibodies against these domains are hypothesized to directly inhibit receptor dimerization and subsequent activation, as opposed to traditional anti-EGFR antibodies which block ligand binding. To accomplish this, peptide mimics of EGFR loops were used as antigens; however, antibodies generated using both yeast surface display and rabbit monoclonal technology were peptide-specific, but did not bind to EGFR protein.(cont) Finally, we developed a mathematical model to describe equilibrium EGFR ligand binding and dimerization. Based on observations that polyclonal antibodies against EGFR domain II or IV eliminate high affinity EGF binding normally observed on the surface of cells, we hypothesized that preformed inactive dimers were the high affinity component. Our model incorporating this hypothesis successfully reproduced experimental data, resulting in characteristic concave-up Scatchard plots.by Ginger Chao.Ph.D

Shedding Light on Ligand Bias in Receptor Tyrosine Kinase Signaling

Receptor Tyrosine Kinases (RTKs) are the second largest family of membrane proteins after the G-protein Coupled Receptors (GPCRs). They consist of a soluble extracellular domain, a single pass transmembrane helix, and an intracellular kinase domain. RTKs are known to oligomerize in the plasma membrane and become signaling competent in the presence of ligand. Ligand bias is the ability of one ligand to preferentially activate one pathway over another. Biased ligands could provide the key to developing many therapeutics that efficiently inhibit or activate specific pathways without dangerous side effects. Here, we investigate the presence and mechanism of ligand bias in RTK signaling. Ligand bias has been extensively studied in GPCRs and many analysis methods have been developed specifically for the GPCR field. We investigate ligand bias as it relates to RTK signaling. Our hope is that understanding ligand bias can help guide therapeutic development. In chapter 1, we introduce the structure-function relationship in RTKs and their ligands. In chapter 2, we research GPCR analytical methods for the assessment of ligand bias and determine which are applicable to RTK signaling. We use real and simulated data to prove the relationship between these methods and to illustrate our prescribed method. In chapter 3, we obtain western blotting data and use the analysis methods from chapter 2 to prove the existence of ligand bias in FGFR1 signaling. We then seek to understand the mechanism of ligand bias in RTKs. It is understood that the mechanism behind ligand bias in GPCRs is that ligand identity contributes to the GPCR conformation. This change in GPCR conformation can affect which substrates the GPCR can interact with. In chapter 3, we show that ligand identity can affect dimer conformation and therefore, we hypothesize that the mechanism may be similar to that of GPCRs. In chapter 4, we use existing knowledge of RTK signaling as well as published crystal structures to propose that RTKs can adapt multiple kinase dimer conformations and that these conformations can be affected by the ligand identity. In chapter 5, we use computational methods including disorder prediction, normal modes analysis, and docking software to show the feasibility of multiple kinase dimer conformations. Finally, in chapter 6, we propose a new analysis method for the assessment of bias, not only for RTKs, but for GPCRs as well, by building on our previous work. In summary, we show that ligand bias exists in RTKs using both qualitative and quantitative analysis methods. Using both FRET and computational methods, we show that the mechanism of ligand bias in RTKs is likely due to differences in kinase dimer conformations. Finally, we propose a new method for analysis of ligand bias that is not exclusive to RTKs that provides a more sensitive detection of ligand bias for future drug design